4. MATERIALS AND METHODS
4.1 Materials
4.1.9 FACS staining panel for B cell kinetic study
Antibody Conjugation Clone Per 100ul Provider
Rat anti-CD45 AF 700 30F11 1ul BioLegend, San Diego, CA, USA
Rat anti-CD19 FITC 6D5 1ul BioLegend, San Diego, CA, USA
Rat anti-CD93 PE AA4.1 2ul eBioscience, San Diego, CA, USA Rat anti-CD23 PE-Cy7 B3B4 1ul eBioscience, San Diego, CA, USA
Rat anti-CD21 PB 4E3 1ul eBioscience, San Diego, CA, USA
Rat anti-CD138 V605 281-2 0.5ul BioLegend, San Diego, CA, USA Rat anti-IgD APC-Cy7 11-26C 1ul eBioscience, San Diego, CA, USA Rat anti-IgM PerCP-Cy5.5 RMM-1 1ul BioLegend, San Diego, CA, USA Rat anti-CD3ε APC 17A2 1ul eBioscience, San Diego, CA, USA Rat anti-CD11b APC M1/70 1ul eBioscience, San Diego, CA, USA Rat anti-Gr1 APC RM6-8C5 1ul (1:40
predilution) DRFZ, Berlin, Germany
24 4.1.10 Antibodies for immunofluorescent microscopy
Antibody Clone Dilution Provider
Primary antibodies
Goat anti-CXCL 13 AF470 1:50 R&D, Minneapolis, MN, USA
Mouse anti-NeuN A60 1:200 Merck, Darmstadt, Germany
Rabbit anti-CD3 Polyclonal 1:200 Abcam, Cambridge, UK Rabbit anti-collagen type I Polyclonal 1:200 Merck, Darmstadt, Germany Rabbit anti-laminin 1+2 Polyclonal 1:400 Abcam, Cambridge, UK Rabbit anti-Ki67 Polyclonal 1:200 Abcam, Cambridge, UK
Rat anti-CD4 L3T4 1:200 BD Biosciences, Franklin Lakes, NJ, USA
Rat anti-CD140b PDGF Receptor b)
APB5 1:200 eBioscience, San Diego, CA, USA
Rat anti-CD13 ER-BMDM1 1:200 Bio-Rad, Puchheim, Germany?
Rat anti-CD68 FA-11 1:200 Bio-Rad, Puchheim, Germany
Rat anti-CD45RO/B220, Alexa Fluor® 488 conjugated
RA3-6B2 1:200 BioLegend, San Diego, CA, USA
Secondary antibodies
Donkey anti-goat, Alexa Fluor® 594 1:400 Life Technologies, Darmstadt, Germany Goat anti-rat, Alexa Fluor® 488 1:400 Life Technologies, Darmstadt, Germany Goat anti-rat, Alexa Fluor® 594 1:400 Life Technologies, Darmstadt, Germany Goat anti-rabbit, Alexa Fluor® 488 1:400 Life Technologies, Darmstadt, Germany Goat anti-rabbit, Alexa Fluor® 568 1:400 Life Technologies, Darmstadt, Germany Goat anti-rabbit, Alexa Fluor® 594 1:400 Life Technologies, Darmstadt, Germany Goat anti-mouse Oregon Green® 488 1:400 ThermoFisher, Waltham, MA USA
4.1.11 Commercial kits
Name Provider
Anti-Mouse/Rat Foxp3 Staining Set PE eBioscience, San Diego, CA, USA
FITC BrdU Flow Kit BD Biosciences, Franklin Lakes, NJ, USA
Mouse CXCL13/BLC/BCA-1 DuoSet ELISA R&D, Minneapolis, MN, USA
Mouse on Mouse (M.O.M. ™) Fluorescein Kit Vector Laboratories, Burlingame, CA, USA
25 4.1.12 Laboratory consumables
Name Provider
0.19±0.01 mm diameter silicon-rubber-coated
monofilament Doccol Corporation, MA, USA
100-µm, 70-µm, 40-µm cell strainer BD Falcon, Franklin Lakes, NJ, USA 14ml BD FalconTM round-bottom tube, snap cap BD Falcon, Franklin Lakes, NJ, USA 15ml, 50ml BD FalconTM tube, dome-seal screw cap BD Falcon, Franklin Lakes, NJ, USA
1ml, 2ml, 5ml syringe BD Discardit, Franklin Lakes, NJ, USA
35x10 (mm) tissue culture dish BD Falcon, Franklin Lakes, NJ, USA 48-well cell culture plate Falcon (Franklin Lakes, NJ, USA) 5ml, 10ml, 25ml BD Falcon™ serological pipets,
individually wrapped BD Falcon, Franklin Lakes, NJ, USA
BD FalconTM 12x75(mm) tube with and without cell
strainer cap BD Falcon, Franklin Lakes, NJ, USA
BD Vacutainer® spray-coated K2EDTA tube, 13x75
(mm) BD Biosciences, Franklin Lakes, NJ, USA
BD Vacutainer® Plus Plastic Serum Tubes,
13x75 (mm) BD Biosciences, Franklin Lakes, NJ, USA
CountBright™ Absolute Counting Beads, for flow
cytometry ThermoFisher Scientific Inc., Waltham, MA USA
Fuchs-Rosenthal chamber
(depth 0.200mm, area 0.0625mm2)
Paul Marienfeld GmbH & Co. KG;
Lauda-Königshofen, Germany Eppendorf® Safe-Lock microcentrifuge tubes, 1.5 mL Eppendorf, Köln, Germany LightCycler® Capillaries (20ul) Roche, Mannheim, Germany Low Profile 819 Disposable Blades Leica, Wetzlar, Germany
Microscope slides (SuperFrost Plus) R. Langenbrinck, Emmendingen, Germany
Pur-Zellin cotton pads Paul Hartmann AG, Heidenheim, Germany
Sterile scalpel #21 B.Braun AESCULAP, Tuttlingen, Germany
Stainless Steel Beads, 5 mm QIAGEN GmbH, Hilden, Germany
Transfer pipette 3.5ml, individually wrapped, sterile Sarstedt AG & Co, Nümbrecht, Germany
26 4.1.13 Laboratory equipment
Name Provider
1H (300 MHz) mouse head surface radiofrequency
(RF) coil RAPID Biomedical GmbH, Rimpar, Germany
20 mm diameter quadrature transmit/receive mouse
head volume resonator RAPID Biomedical GmbH, Rimpar, Germany
7T Bruker PharmaScan 70/16 magnet Bruker BioSpin, Ettlingen, Germany
CasyTon cell counter Casy-Technology, Innovatis AG, Reutlingen, Germany
CatWalkTM XT system Noldus Information Technology, Wageningen, The Netherlands
Centrifuge, Universal 30 RF Thermo Electron, Oberhausen, Germany
Centrifuge 5804 R Eppendorf, Köln, Germany
Electrophoresis chamber Criterion Bio-Rad, Munich, Germany Fluorescence microscope, DMRA2 Leica, Wetzlar, Germany
Eppendorf 5810R centrifuge Eppendorf, Köln, Germany
Fluorescence microscope, DMI 6000B Leica, Wetzlar, Germany
FACSCanto II flow cytometer BD Pharmingen, Franklin Lakes, NJ, USA Homoeothermic blanket system Harvard Apparatus, Holliston, MA, USA LC Carousal centrifuge 2.0 Roche Diagnostics, Indianapolis, IN, USA
Lightcycler 2.0 Roche, Mannheim, Germany
Leica confocal microscope Leica TCS SPE, Nussloch, Germany
LSR II flow cytometer BD Pharmingen, Franklin Lakes, NJ, USA
Mastercycler gradient Eppendorf , Köln, Germany
Microtome MICROM HM 330 Thermo Scientific, Waldorf, Germany Mini Rocking Platform, Biometra WT 17 Biometra GmbH, Göttingen, Germany NanoDrop ND2000 spectrophotometer Thermo Scientific, Asheville, NC, USA
pH 522 pH-meter Wissenschaftlich-Technische Werkstätten GmbH,
Weilheim, Germany
SpectraMax 190 Microplate Reader Molecular Devices, LLC., Sunnyvale, CA, USA
Recovery box MediHEAT PecoServives Ltd. Brough, Cumbria,
UK
RET-3 rectal probe for mice Physitemp Instruments Inc., Clifton, NJ, USA Rodent Brain Matrix – Mouse, Adult 30g,
Coronal Sections ASI Instruments, Inc., Warren, MI, USA
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Sliding microtome Leica SM2000R, Nussloch, Germany
Thermomixer 5436 heating block Eppendorf Vertrieb Deutschland GmbH, Hamburg, Germany
TissueLyser LT QIAGEN GmbH, Hilden, Germany
TissueLyser Bead Dispensers QIAGEN GmbH, Hilden, Germany
Vet ABC Animal blood counter scil animal care company GmbH, Viernheim, Germany
4.1.14 Analytical software
Name Provider
Bruker Paravision 6.0 Bruker BioSpin, Ettlingen, Germany
CasyTon cell counter and analyser system Innovatis AG, Reutlingen, Germany
FACSDiva 6.1.3 BD Pharmingen, Franklin Lakes, NJ, USA
FlowJo 10.0.8r1 Tree Star, Ashland, OR, USA
ImageJ ImageJ 1.42q Wayne Rasband, NIH, Bethesda,
USA
LightCycler Software (build 4.1.1.21) Roche Molecular Biochemicals, Basel, Switzerland
LEICA LAS AF Lite Leica, Wetzlar, Germany
Mayo Clinic Analyze 5.0 Biomedical Imaging Resource, Analyze Direct, Overland Park, KS, USA
Noldus software for CatWalk Noldus Information Technology, Wageningen, The Netherlands
Stereo Investigator software® MBF Bioscience, VT, USA
GraphPad Prism 6.0 GraphPad, San Diego, CA, USA
IBM SPSS Statistics 25 for Windows IBM Somers, NY, USA
28 mouse tunnel and igloo (Plexx BV). Mice had ad libitum access to food (standard chow) and water (where study design indicates, water was replaced with an antibiotic solution). After MCAo surgery, enrichment was removed and for the first three post-operative days soft pellet food (standard chow softened with water/antibiotic solution) was given in addition. All animal experiments were conducted in accordance with the European Community Council Directives 86/609/EEC and German national laws and approved by the local authority (Landesamt für Gesundheit und Soziales, Berlin, Germany).
4.2.1.2 Middle cerebral artery occlusion
MCAo was performed adhering to the standard operating procedures of the laboratory (Engel, Kolodziej et al. 2011). In brief, anesthesia was induced with 2.5% isoflurane (Forene, Abbott) in 1:2 mixtures of O2 / N2O and maintained at 1.0%–1.5% isoflurane. Silicon rubber-coated monofilament with a diameter of 0.19 ± 0.01 mm (Doccol) was introduced into the common carotid artery, advanced along the internal carotid artery towards the origin of the MCA, and either withdrawn immediately to allow instant reperfusion (sham operation) or left there for 60 min. For reperfusion, the inserted filament was withdrawn and the internal carotid artery was ligated in mice under anesthesia. Body temperature was maintained with a heating pad. A drop of 2% Xylocain gel was applied to the wound for pain relief. Success of MCAo was verified using the modified Bederson score (Bederson, Pitts et al. 1986). Animals were randomly allocated into different operators. After surgery, animals were allowed to recover in a heated cage before returning to their home cages.
Animals with unsuccessful stroke, as confirmed by MRI assessment or death on the day of operation, were excluded.
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4.2.1.3 T2-weighted Magnetic Resonance Imaging (MRI) for infarct size determination
To quantify the ischemic lesion, animals were subjected to a T2-weighted MRI at 24 h and 7 days after MCAo. Anesthesia was induced at 2.0% isoflurane in O2 / N2O mixture (1:2) and maintained at 1.0%-1.5% isoflurane through a vaporizer and delivered through a face mask. Body temperature was kept stable at 36.5±0.5 °C with a heating pad. Respiratory rate was monitored with the Small Animal Monitoring and Gating System. The mouse’s head was fixed in a magnet bore. Axial T2-weighted images were obtained using a 7T Bruker PharmaScan 70/16 magnet with a 20 mm diameter quadratum transmit/receive mouse head volume resonator radiofrequency coil and Bruker Paravision 6.0 software using Rapid Acquisition with Relaxation Enhancement (RARE) sequence (TE 36 ms, TR 4200 ms, FOV 28 mm, 20 contiguous slices with a thickness of 500 µm and interslice distance 500 µm, matrix=256 × 256, field of view=25.6 mm x 25.6 mm, repetition time=4200 ms, echo time=36 ms, RARE factor=8, 6:43 min). Axial slices covered the distance between the olfactory bulb and the cerebellum. Acquired images were analyzed semi-automatically with Mayo Clinic Analyze software version 5.0. The volume difference between the contralateral hemisphere and non-infarcted ipsilateral hemisphere was divided by the volume of the whole ipsilateral hemisphere. The calculated percentage was expressed as edema-corrected infarct size.
Mice with an infarct size less than 5% were excluded from the final analysis.
4.2.1.4 Drug and antibody treatment
To minimize the influence of infection on stroke-induced autoreactivity and outcome parameters (Hetze, Engel et al. 2013), preventive antibiotic marbofloxacin (5mg/kg, diluted in saline to 1mg/ml, prepared fresh daily) was administered 24 h before and until day 6 after MCAo. Alternatively, in some experiments preventative antibiotic enrofloxacin (diluted in drinking water at 0.35 mg/ml) was administered 24 h before and until day 7 after MCAo. In addition, mice received 10 mg/kg of enrofloxacin injected intraperitoneally (i.p.) once a day for the first 3 days after MCAo. On day 3, mice were randomly allocated into either the CD4 depletion (clone GK1.5) group or the isotype control (clone LTF-2) group. On the same day, mice in the CD4 depletion group received intraperitoneal (i.p.) injection of 200ug CD4 depleting antibody (diluted in sterile PBS at 1mg/ml).
Three more injections were administered on days 5, 7 and 9 after MCAo. In total, one mouse received 800ug CD4 depleting antibody to eliminate CD4+ T cells in the blood circulation and
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secondary lymphoid organs. The control group received only the same amount of isotype control antibody (diluted in sterile PBS at 1mg/ml) according to the same injection scheme.
For mice undergoing analysis of cellular proliferation, 100ul 10mg/mL solution of BrdU diluted in sterile 1X DPBS (BD) was injected i.p. into each animal 2 hours before sacrifice.
4.2.1.5 Neurological Deficit Score (De Simoni)
The Neuroscore was performed blindly as described (Orsini, Villa et al. 2012), 3 days before and 3, 7 and 13 days after MCAo or sham surgery when necessary. This test was used to evaluate the general health status and focal neurological malfunction after MCAo. The score ranges from 0 (no deficits) to 56 (representing the worst performance in all items). The Neuroscore results include the following general deficits: fur (0-2), ears (0-2), eyes (0-4), posture (0-4), spontaneous activity (0-4), and epileptic behavior (0-12); and the focal deficits: body symmetry (0-4), gait (0-4), climbing on a surface inclined at 45° (0-4), circulating behavior (0-4), front-limb symmetry (0-4), compulsory circulating behavior (0-4) and whisker sensation to light touch (0-4). The sum of all items was calculated to measure the general and focal deficits of each animal.
4.2.1.6 Catwalk and gait analysis
Quantitative gait analysis is useful for objective assessment of walking ability and exploring new parameters as targets for neuroprotective treatment. CatwalkTM XT is a computer-assisted automated gait analysis system which was originally developed as a tool to enhance assessment of functional outcome in spinal cord injury (SCI) models (Hamers, Koopmans et al. 2006). It has also been reported as a promising tool to assess mid to long-term outcome in experimental stroke (Hetze, Romer et al. 2012). Animals were trained on three consecutive days before baseline measurement.
They acquired a minimum of 3 compliant runs, fulfilling a minimum run duration of 0.5s, maximum run duration of 5s, and a maximum speed variation of 60%. Runs were excluded if an animal turned, and the animal was given another try. A second measurement was carried out at 10 days after MCAo to monitor mid-term recovery. Data was analyzed using the Catwalk XT software according to the manufacturer’s instructions.
Gait parameters can be classified into three main categories (Hamers, Koopmans et al. 2006):
parameters related to (a) individual paws, such as width and length of a paw print, duration of paw
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contact, the pressure caused by a paw, etc. (b) the position of footprints, for instance relative print position, stride length and base of support and (c) the time-dependent relationship between footprints, such as phase dispersion and support formula. A summary of impairments in the right hind limb was designed post hoc. Reductions in maximal contact area, normalized swing speed, stand and stride length of the right hind limb were summed as an overall impairment score. For each item, only reduction got one point.
4.2.1.7 Y maze and memory evaluation
Spatial working memory performance of mice was assessed using the Y maze test (Maurice, Hiramatsu et al. 1994). This test is based on the natural tendency of mice to explore novel environments more than familiar ones. Accordingly, mice with intact working memory alternate among different arms of the maze during subsequent entries. Spontaneous alternation behavior during an 8-minute session was recorded on day -1, day 13 and day 48 after 60 min MCAo. The wooden maze, painted black, consisted of three arms positioned at 120° to each other (arm dimensions: 40cm long, 10.5 cm high and 3 cm wide). The test was performed in a testing room with diffused dim light (35 lux) to provide a less anxiogenic environment. Each mouse was placed at the end of one arm and allowed to move freely throughout the duration of the test. An entry was considered complete when all four limbs crossed a line located 6.5 cm from the arm entrance. The series of arm entries were recorded by video camera (Panasonic, CCTV camera), and alternation was defined as successive entries into three different arms in overlapping triplet sets. The percentage of alternation was calculated as a ratio of performed to total possible alternations multiplied by 100.
Before and after testing each animal, the maze was cleaned thoroughly with Decosept AF to minimize influence of odor trails from previous animal.
Induction of stroke with extensive lesions can reduce mice’s overall locomotor activity, which can be a confounding factor in a memory test that requires motor activity. For this reason, mice that performed less than 12 entries during an 8-minute session were excluded from the final analysis.
4.2.1.8 Multiple collections of blood from living animals
Methods for obtaining blood samples from mice are generally difficult. In order to get multiple blood samples from living animals, submandibular bleeding of mice using a lancet (Golde, Gollobin et al. 2005) was used in this study. The blood collection tubes, either with anti-coagulant
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ethylenediaminetetraacetic acid (EDTA) (blood sample for harvesting leukocytes) or without EDTA (blood sample for harvesting serum), were used to collect approximately 100ul blood. After blood collection, a sterile gauze pad was simply applied with a little compression for less than a minute to stop the bleeding. After release, the mice groomed themselves and within a short time showed no evidence of the blood draw.
Blood was left to clot undisturbed at room temperature. 30 minutes later, the clot was removed by centrifuging at 1000g for 10 minutes in a refrigerated centrifuge. Following centrifugation, the liquid component (serum) was immediately transferred to a clean 1.5ml polypropylene tube. The
Mice were laid on their back and fixed on a board. Body and fur were cleaned with 70% ethanol before abdominal incision. Under sterilized condition, up to 500ul whole blood was collected from the inferior vena cava using a 1ml syringe and placed into a blood collection tube without anticoagulants. Spleen, thymus, lymph nodes (cervical, lumbar and mesenteric) were removed and kept in Phosphate Buffered Saline (PBS, dilute 10x stock solution with sterilized water, GIBCOTM) on ice.
Each animal was then transcardially perfused with 20ml of ice-cold PBS at 80cm water height. After careful removal of the skull, the whole brain was dissected without the cerebellum and kept in ice-cold complete Roswell Park Memorial Institute (RPMI) 1640 medium (medium supplemented with 10% fetal calf serum (FCS), 50U/ml penicillin, 50ug/ml streptomycin, 2mM L-alanyl-L-glutamine).
The tissue was processed immediately. For the experiment designed for immunohistochemistry, perfusion was first done with 20ml ice-cold saline, followed by 20ml ice-cold 4% paraformaldehyde (PFA) in 0.1 M Sörensen buffer (consisting of 80 mM disodium phosphate, 20 mM sodium dihydrogen phosphate monohydrate in distilled water; pH 7.4) per animal. Brains were collected and post-fixed overnight in 4% PFA. After a short rinse in PBS, the fixed brains were transferred to 30%
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D (+)-sucrose solution in 0.1M Sörensen buffer for cryoprotection. After 3 days, when the brains had totally sunk to the bottom, they were snap-frozen using 2-methylbutan (Carl Roth) and dry ice.
4.2.2 Molecular, immunological and immunohistochemistry methods 4.2.2.1 Isolation of leukocytes from spleen, lymph node, brain and blood
Brains, stored in complete RPMI 1640 medium, were transferred to 35 mm-diameter tissue culture dishes containing the same medium. Brains were separated into the ipsilateral and contralateral hemispheres using a sterile scalpel. Each hemisphere was pressed through a 70 μm pore-size cell strainer into a 50 ml Falcon tube filled with complete RPMI 1640 medium using a 2-ml syringe stamp. The same procedure was repeated until the medium, refilled in strainer, became clear. Cell suspension was washed once with complete RPMI 1640 medium by centrifugation at 1500 rpm at room temperature (RT) for 10 min. 6 ml single-cell suspension with 35% Easycoll gradient prepared in complete RPMI 1640 medium was cautiously pipetted using only gravity onto 4 ml 70% Easycoll gradient. After centrifugation at 2400 rpm and RT without brake for 30 min, the interface with mononuclear cells (MNCs) between two gradients was isolated. After washing isolated cells once again with complete medium, a small portion of MNCs were stained with Trypan Blue dye and counted under a light microscope using a Fuchs-Rosenthal chamber.
Spleens and lymph nodes were forced through a 100 μm pore-size cell strainer with a 2-ml syringe stamp and washed once with complete RPMI 1640 medium. Erythrocytes in the spleen were lysed during 2 min incubation of cell pellets with RBC lysis solution and occasional shaking with fingers.
After resuspension with complete medium, splenocytes and lymphocytes were pipetted through a 40 μm pore-size cell strainer and washed once again. Splenocytes were quantified with automated CasyTon cell counter (with a program configured for counting mice splenocytes). Lymphocytes from lymph nodes were counted using the Trypan Blue method described above.
The peripheral blood was collected either by submandibular bleeding, as described, (Golde, Gollobin et al. 2005) from living animals (baseline and any intermediate time points) or from the inferior vena cava in deeply anesthetized mice at the endpoint. In both cases, whole blood was immediately collected into K2EDTA-coated blood collection tubes with occasional shaking to prevent coagulation and placed at RT until FACS staining on the same day. All cells were washed
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once again with FACS buffer containing 2% FCS in sterile PBS before following staining procedure for flow cytometry.
4.2.2.2 FACS analysis
4.2.2.2.1 FACS analysis of peripheral blood for phenotyping 2D2 mice
Blood samples from 2D2 mice were collected into EDTA-coated tubes and transferred at RT from the animal facility to the lab. Phenotyping using FACS was done on the same day. Erythrocytes were lysed with 1ml 1x Pharm Lyse lysis buffer (diluted in distilled water) per 50 ul blood sample using vortex. After 10 min of incubation at RT, samples were centrifuged at 1500rpm at RT for 5 min. After removal of supernatant, cell pellets were resuspended in 200 ul FACS buffer (PBS with 2% FCS + 15.4 mM sodium azide) and transferred to a new FACS tube filled with 2 ml FACS buffer. After centrifugation at 1500 rpm at 4°C for 5 min and removal of supernatants, blood leukocytes were stained with 50 ul 1:100 mixture of monoclonal antibodies containing APC conjugated anti-CD4 and PE conjugated anti-TCR Vβ11 (detecting MOG TCR transgenic CD4+ T cells). Antibodies were incubated on ice for 30 min with light protection. Isotype control was stained with APC-rat IgG2α κ chain and PE-rat IgG2β κ chain. Samples were washed once again with FACS buffer and centrifuged to remove unbound antibodies. Final acquisition was done on FACSCanto II flow cytometer and analyzed with FlowJo software V10. Animals with over 80% of CD4+ T cells positively expressing TCR Vβ11 were considered transgenic 2D2 mice and allowed into further experiments.
4.2.2.2.2 FACS analysis of leukocytes proliferation by flow cytometry
106 BrdU-pulsed splenocytes, thymocytes, leukocytes from lymph nodes and whole brain MNCs from each hemisphere were added to 12x75-mm FACS tubes, washed in FACS buffer and
106 BrdU-pulsed splenocytes, thymocytes, leukocytes from lymph nodes and whole brain MNCs from each hemisphere were added to 12x75-mm FACS tubes, washed in FACS buffer and